Project Summary/Abstract: Alterations in the immune system occur with aging, likely contributing to the development of infections and malignancies. Although the latter finding suggests an age-associated decline of the immune system, aging could be regarded as a condition with dysregulated inflammation (inflammaging). In T cell immunity, probably, the most prominent change with aging is the expansion of memory CD8+ T cells in peripheral blood although its exact mechanism(s) and significance are yet to be determined. My lab investigated whether the expansion of human memory CD8+ T cells with aging was secondary to increased expression of IL-7 receptor alpha chain (IL-7R?) which dictates the response to the pro-survival cytokine IL-7. We found two cell populations which expressed low and high levels of IL-7R? (IL-7R?low and high) in effector memory (EM) CD8+ T cells in peripheral blood. Of interest, older adults (OA, age ? 65) had expansion of IL- 7R?low EM CD8+ T cells (up to 70% of total CD8+ T cells), which are largely cytotoxic and inflammatory cytokine producing cells, compared to young adults (YA, age ? 40). Such cell expansion is associated with cytomegalovirus infection, which persists for lifetime, as well as with enhanced production of and proliferative response to IL-15 that promotes the maintenance and effector function of memory CD8+ T cells. However, the significance of the expansion of IL-7R?low EM CD8+ T cells in OA remains largely unknown, especially in the context of inflammation. To address this critical question, we performed a genome-wide DNA methylation analysis in IL-7R?low and high EM CD8+ T cells since DNA methylation modulates gene expression. IL-7R?low EM CD8+ T cells have hypomethylation of DNA in a group of chemotaxis-associated genes, including CX3CR1 (receptor for fractalkine), with enhanced expression. Plus, we noticed increased CX3CR1 expression by IL- 7R?low EM CD8+ T cells in OA compared to YA as well as the effect of aging on DNA methylation of the CX3CR1 promoter in the same cells. Thus, we hypothesize that aging ?qualitatively? enhances the pro- inflammatory traits of human IL-7R?low EM CD8+ T cells by affecting gene regulatory mechanisms, including DNA methylation and IL-15-mediated responses in addition to expanding them and that soluble factors produced from such cells promote inflammation by activating other cells. This hypothesis will be tested with: Aim 1. Investigate whether aging promotes IL-7R?low EM CD8+ T cell-mediated inflammatory response in humans by altering inflammatory gene regulation via DNA methylation and transcription factors; Aim 2. Elucidate age-associated alterations in IL-15-mediated inflammatory response in human IL-7R?low EM CD8+ T cells; and Aim 3. Investigate the significance of age-associated alterations of IL-7R?low EM CD8+ T cells in promoting inflammatory responses using ex vivo bioassay and humanized mice. Our results will shed light on the biological significance of expanded memory CD8+ T cells with aging, especially in the aspect of inflammation, as well as on the possible role of gene regulations in promoting this process.